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    AST SOP

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    humanelaboratory

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    AST SOP

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    AST SOP

    Uploaded by

    humanelaboratory

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    © All Rights Reserved
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    of 6

    SOP

    HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY


    TITTLE: Standard Operating Procedure SOP No: HGL/0017
    (SOP) For Antimicrobial Version: 1
    Susceptibility Testing
    Date Issued: 04/10/2023 Page 1 of 1 Next Review Date:

    1.0 Purpose:

    The purpose of this standard operating procedure is to explain in details the


    procedures for antimicrobial susceptibility testing method.

    2.0 Responsibility:

    This SOP is initiated and reviewed by the Humane Laboratory staffs and
    completed by Laboratory Manager and laboratory consultant who have been
    well trained on all information listed under “Prerequisite Training Materials” and
    this SOP with the scope/view of carrying out effective and efficient antimicrobial
    susceptibility testing

    3.0 Sample Acceptance Criteria

    Properly collected sample, accompanied with completed Laboratory request


    form and evidence of payment. Well labeled, uncontaminated and moist in the
    case of swap which supports viable organisms

    4.0 Rejection Criteria

    Incompletely or incorrectly labeled, contaminated sample and when swaps are


    allowed to stay for a long period of time unmoist

    5.0 Turn Around Time

    72 hours
    SOP

    HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY


    TITTLE: Standard Operating Procedure SOP No: HGL/0017
    (SOP) For Antimicrobial Version: 1
    Susceptibility Testing
    Date Issued: 04/10/2023 Page 1 of 2 Next Review Date:

    6.0 MEDIA PREPARATION


    6.1 Materials:
    6.1.1 Media powder
    6.1.2 Weighing balance,
    6.1.3 Measuring Spoon,
    6.1.4 Foil paper or weighing pot,
    6.1.5 Conical flask,
    6.1.6 Distilled water,
    6.1.7 Hot plate stirrer,
    6.1.8 Autoclave,

    6.2 Procedure:
    6.2.1 cleaning/Disinfection work bench
    6.2.2 Spray work bench with freshly prepared 1% hypochloride (bleach) solution.
    Clean using dry cotton wool.
    6.3 Muller Hilton Agar Media preparation:
    6.3.1.Weigh the required quantity of the media powder for specific volume in a
    beaker according to the instructions of the manufacturer as given on the
    dehydrated media bottle or media preparation sheet
    6.2.3 Place the media powder in a conical flask of graduation that is twice the
    required volume of the media and add appropriate quantity of sterile distilled
    water and mix it properly
    6.2.4 Dissolve the media by heating with continuous stirring until completely
    dissolved on the hot plate .
    6.2.5 Adjust the pH with 0.1N HCl/0.1N NaOH (only if necessary or as instructed)
    6.2.6 Seal the mouth of the conical flask using corresponding bottle cover or cotton
    plugs
    6.2.7 Unless specified otherwise, Sterilize at 121°C /15psi for 15 minutes.

    SOP

    HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY


    TITTLE: Standard Operating Procedure SOP No: HGL/0017
    (SOP) For Antimicrobial Version: 1
    Susceptibility Testing
    Date Issued: 04/10/2023 Page 1 of 3 Next Review Date:

    6.2.8 Pour the prepared media aseptically (preferably in a biosafety cabinet) into
    petri-dishes, label the solidified media and keep/store at a temperature of 2 -
    8 0C
    Note:- always check for media sterility before storage and ensure that stored media
    do not exceed three weeks

    7.0 PREPARATION OF INOCULUM


    7.1 Materials
    7.1.1 Wire loop
    7.1.2 UV VIS-Spectrometer
    7.1.3 Isolated colonies
    7.1.4 Saline solution
    7.1.5 Test tubes
    7.2 Procedure

    7.2.1 Use a sterile loop to pick colonies from an overnight culture on non-selective
    media such as tryptic soy agr, nutrient agar e.t.c. Use several similar
    morphological colonies (when possible) to avoid selecting an atypical variant
    7.2.2 Suspend the colonies in saline and mix to an even turbidity.
    7.2.3 Adjust the density of the organism suspension by adding saline or more
    bacteria to give an absorbance of 0.08 to 0.13 at 620 nm which is equivalent
    to 0.5 McFarland
    7.2.4 Ensure that whole preparation and inoculation of 0.5 or 1.0 McFarland
    standard does not exceed 15 minute
    Note:- Different bacterial isolate may require different density/turbidity, e.g E coli
    requires 0.5 McFarland standards while other bacterial isolate such as

    SOP

    HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY


    TITTLE: Standard Operating Procedure SOP No: HGL/0017
    (SOP) For Antimicrobial Version: 1
    Susceptibility Testing
    Date Issued: 04/10/2023 Page 1 of 4 Next Review Date:

    streptococcus pneumonia may requires 1.0 McFarland standard especially when it is


    suspended from chocolate agar
    8.0 INOCULATION OF AGAR PLATES
    8.1 Materials:
    8.1.1 Sterile swab stick,
    8.1.2 forceps,
    8.1.3 Media (most suitably, Mueller Hilton Agar),
    8.1.4 Antibiotic discs

    8.2 Procedure:

    8.2.1 Make sure that agar plates are at room temperature prior to inoculation.
    8.2.2 Dip a sterile cotton swab into the suspension 0.5 or 1.0 McFarland standard
    depending on the bacterial isolate
    8.2.3 To avoid heavy inoculum growth of Gram-negative bacteria, remove excess
    fluid by pressing and turning the swab against the inside of the tube.
    8.2.4 For Gram-positive bacteria, do not press or turn the swab against the inside of
    the tube especially if using 0.5 McFarland turbidity
    8.2.5 Smear the dipped swap all over the media (ensure no gaps between streaks)
    8.2.6 Allow disks to reach room temperature before opening cartridges or
    containers used for disk storage.
    8.2.7 Apply disks firmly to the surface of the inoculated media plate within 15
    minutes of inoculation.
    8.2.8 Place the antibiotic discs at adequate spacing (at least 2cm apart) with some
    sterile fine pointed forceps
    8.2.9 Invert agar plates and make sure disks do not fall off the media surface.

    SOP

    HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY


    TITTLE: Standard Operating Procedure SOP No: HGL/0017
    (SOP) For Antimicrobial Version: 1
    Susceptibility Testing
    Date Issued: 04/10/2023 Page 1 of 5 Next Review Date:

    8.2.10 Incubate plates within 15 min1 of disk application


    8.2.11 Incubate in an incubator for 18 - 24 hours
    8.2.12 Measure the inhibition zone diameters to the nearest millimetre with a ruler
    or vernier calliper.

    9.0 DIRECT SUSCEPTIBILITY TEST (Disk diffusion method)


    9.1 Materials:
    9.1.1 Sterile swab stick,
    9.1.2 forceps,
    9.1.3 Media (most suitably, Mueller Hilton Agar)
    9.1.4 Antibiotic discs
    9.2 Procedure:
    9.2.1 Make sure that agar plates are at room temperature prior to inoculation
    9.2.2 Hold the the tissue with the flamed forceps and use sterile swab stick to
    gently macerate the tissue to ensure a good tissue representation is achieved
    9.2.3 Using the swap inoculate the tissue all over the surface of Mueller Hilton Agar
    9.2.4 Incubate for 3 - 4 hours in peptone broth for swabbed samples and then
    Inoculate precisely on Mueller Hilton Agar by spreading all over it surface
    9.2.5 Place the antibiotic discs at adequate spacing (2cm apart) with a sterile fine
    pointed forceps and press gently to ensure contact with the medium.
    9.2.6 incubate for 18 to 24 hours at 370C
    10.0 RESULT:
    Use vernier calipers / ruler to read the diameter of the zones of inhibition as follows
    10.1 SENSITIVE; Diameter of zone of inhibition around the disc is greater than or
    equal to 6mm

    SOP

    HUMANE VETERINARY DIAGNOSTIC AND NUTRITIONAL LABORATORY


    TITTLE: Standard Operating Procedure SOP No: HGL/0017
    (SOP) For Antimicrobial Version: 1
    Susceptibility Testing
    Date Issued: 04/10/2023 Page 1 of 6 Next Review Date:

    10.2 INTERMEDIATE or INCREASED DOSE; Diameter of zone of inhibition around the


    disc is less than 6mm greater than 3mm
    10.3 RESISTANT; Diameter of zone of inhibition around the disc is less than 2mm
    Note:- the zones of inhibitions is read accordingly except for few antibiotics that
    have slower or faster rate of diffusion but the result above are in
    approximate to most antibiotics

    11.0 REFERENCES
    11.1 http://www.eucast.org.
    11.2 http://bsac.org.uk/susceptibility/template-program

    12 . Revision History

    Revision Author Revisions Made Effected Date


    00 Henry Hosea Carl 04/10/2023
    01 Mal. Usman Auwal M. 04/10/2023
    02 Dr Mustapha H. H. 04/10/2023
    03 Abdullahi Suleiman 04/10/2023
    04 Dr Rahila Haruna 04/10/2023

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